The ubiquitination of CD274 at MERCs enhances the anti-tumor immunity of cervical cancer

Mitochondria-ER contact sites (MERCs) are crucial signaling hubs, but their role in anti-tumor immunity is unclear. This study revealed that the mitophagy regulator PRKN ubiquitinated CD274 at MERCs in human cervical cancer cells, a key mechanism for anti-tumor immunity. CD274 expression inversely correlated with PRKN in cervical cancer. Upon mitophagy activation, CD274 was recruited from ER to MERCs by PINK1, enhancing its interaction with PRKN. PRKN then ubiquitinated CD274 at residues K89 and K105 within its extracellular domain. Functionally, a ubiquitination-deficient CD274 mutant promoted anaerobic glycolysis and MTOR signaling, accelerating cancer cell growth. Coculture with ubiquitination-deficient CD274 mutant-expressing cancer cells increased the CD8+ T-cells’ exhaustion. Single-cell RNA sequencing of mouse tumors showed the expansion of the exhausted CD8+ T cells and myeloid-derived suppressor cells (MDSCs) with ubiquitination-deficient CD274 mutation. In vivo, a ubiquitination-deficient CD274 mutant accelerated tumor growth and reduced the therapy efficacy of immune checkpoint inhibitors. Conversely, clinical sample analysis showed that CD274 localization at MERCs or its ubiquitination levels were closely associated with the improved immunotherapy efficacy. Thus, mitophagy-dependent recruitment of CD274 to MERCs for PRKN-mediated ubiquitination is a novel pathway that activates the anti-tumor immunity and improves the immunotherapy efficacy, presenting a promising strategic target for cervical cancer treatment.Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; CD, cluster of differentiation; CHX, cycloheximide; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GZMB, granzyme B; IFNG, interferon gamma; LDHA, lactate dehygrogenase A; MAP1LC3, microtubule-associated protein 1 light chain 3; MFN2, mitofusin 2; MHC, major histocompatibility complex; MTOR, mechanistic target of rapamycin kinase; OCR, oxygen consumption rate; PBMC, peripheral blood mononuclear cell; PDCD1, programmed cell death 1; PI, propidium iodide; PINK1, PTEN induced putative kinase 1; PKM, pyruvate kinase, muscle; RPS6, ribosomal protein S6; TNF, tumor necrosis factor; TME, tumor microenvironment.